1. Field of the Invention
This invention relates to solid phase immunoassays using immobilized immunoreagents and more particularly to elements for solid phase immunoassay comprising immunoreagents immobilized on carriers made of inert synthetic resins. The invention also relates to methods of preparing synthetic resin surfaces for use as carriers for immunoreagents.
2. Description of the Prior Art
Immunoassay using immobilized immunoreagents is an analytical method widely used in biochemical analysis. In the conventional procedure an immunoreagent, e.g., an antigen or antibody, is first immobilized on the surface of an analytical element, e.g., a test tube, a rod or stick, beads of glass or plastic, or the like. The immobilized immunoreagent is then contacted with an analyte solution containing a complementary immunoreagent, whereby an immobilized immunocomplex is formed. The immobilized immunocomplex can then be easily separated from the unreacted analyte solution, e.g., by simply removing the analyte solution by aspiration, decantation, or the like, preferably with repeated washing of the immobilized immunocomplex. The separated immobilized immunocomplex may then be subjected to further processing to quantitate the amount of immunocomplex. For example, in a radioimmunoassay, the amount of adsorbed complex may be determined by counting radioactive disintegrations; in an enzyme-linked immunosorbent assay (ELISA) the adsorbed complex which has an enzyme coupled thereto, is contacted with a substrate for the enzyme to produce a detectable product; in an immunofluorescent assay, the fluorescent intensity of a fluorescent substance linked to the immunocomplex may be measured, or the like.
Among the many different materials which have been used as carriers for an immunoreagent are glass, metal, and various plastics such as polystyrene, polyvinyl chloride, silicone resins, polyethylene and the like. In some cases the immunoreagent has been immobilized by covalent bonding to the carrier, while in other cases adsorption of the immunoreagent has proved adequate. Synthetic resin carriers have been widely used because of their economy and convenience and ease of handling, but problems remain in immobilizing enough immunoreagent on their surfaces for maximum sensitivity of the immunoassay, especially when the reagent is merely adsorbed onto the surface. Furthermore, the synthetic resin carriers used previously are inadequate for some of the newer immunoassay techniques such as thermochemiluminescent immunoassay, which requires that the carrier with immobilized immunocomplex be heated to relatively high temperatures, i.e., 200.degree. C. to 300.degree. C. The conventional plastic carriers cannot be used under these conditions because they soften or even melt at such temperatures.
On the other hand, certain plastics are known which can be used at temperatures of 200.degree. to 300.degree. C. without melting or deformation, for example, polyimide synthetic resins, and polytetrafluoroethylene (PTFE) and related fluorinated olefin polymers. However, these resins are very inert and non-adhesive, and it has been thought that satisfactory adsorption of immunoreagents to such surfaces was not possible.
Prior workers in this field do not appear to have attempted to use a polyimide as a carrier for an immunoreagent in a solid phase immunoassay.
Some attempts have been made to use PTFE as a carrier for solid phase immunoassay, but the procedures have been unsuccessful or have had serious drawbacks.
Shekarchi, et al., J. Clin. Microbiology 16(6), 1012-1018 (December, 1982) disclose an immunoassay procedure wherein an immunoreagent is immobilized on a small stick, i.e., "microstick", for easy manipulation of the reagent and the immunocomplex. While a number of materials were investigated for use in such microsticks, including stainless steel, nylon, polycarbonate, polystyrene and PTFE, it was found that the PTFE, cleaned by the conventional procedure of rinsing with 6N HCl, adsorbed very little of the immunoreagent as compared with the other materials and could not be used as a base for the immunoreagent until it had been coated with polycarbonate or nitrocellulose.
German Offenlegungsschrift No. 32 00 822, published July 21, 1983, discloses a method for activating the surface of PTFE articles, in order to bond immunoreagents covalently, by contacting the PTFE surface with an ammoniacal solution of sodium, followed by treatment with carbodiimide. The process was apparently attempted because it was found that adsorption of the immunoreagent on PTFE was unsatisfactory. This process is complex and uses reagents which are difficult to handle and even dangerous. Furthermore, there is some question whether the procedure of this German application actually can immobilize a useful amount of immunoreagent on PTFE.
Hence, a need has continued to exist for improved immunoassay procedures using immunoreagents immobilized on inert carriers such as polyimides and fluorinated polymers and for a practical method of immobilizing immunoreagents on the surfaces of such synthetic resins.